Transformation of cultured plant cells using recombinant Agrobacterium, followed by regeneration of the transformed cells into whole plants, has become the standard means for producing transgenic plants. Various methods employing Agrobacterium vectors have been developed and are known in the art, including the binary plasmid system disclosed in U.S. Pat. No. 4,940,838 (Schilperoort, et al., Jul. 10, 1990), and the so-called co-integrate plasmid system disclosed in U.S. Pat. No. 4,693,976 (Schilperoort, et al., Sep. 15, 1987). Most current methods employ disarmed Agrobacterium, that is, Agrobacterium that has had the tumor-inducing functions deleted or inactivated, so that transformation does not cause tumorous growth, but rather permits growth of normal tissue callus capable of being regenerated into a normal plant.
The use of Agrobacterium, as with most other transformation techniques, has a drawback. In order to obtain a plant that is uniformly transformed (that is, has the heterologous DNA present in every cell) it is necessary to transform individual cells and regenerate therefrom a somatic clone. The cells of some plant species are not easily maintained in tissue culture, and are not easily regenerated into somatic clones. One technique that has been investigated to overcome these obstacles is the use of pollen as a vector. By transforming pollen, then using the transgenic pollen to fertilize a receptive plant, transgenic seed containing the heterologous DNA can be produced. The transgenic seed can be germinated to naturally produce a transgenic plant.
The terms "transgenic pollen" or "transformed pollen" as used in connection with the present invention are defined as Agrobacterium-treated pollen or germinated pollen that is capable of delivering DNA, whether within the pollen or germinated pollen or within an Agrobacterium that is associated with the pollen tube, to the ovum. While not wishing to be bound by theory, there are at least two possible mechanisms by which the treated germinated pollen (i.e. "transgenic pollen" or "transformed pollen" as the term is used hereinafter) of the present invention could be delivering the heterologous DNA to the plant ovum. One possible mechanism involves the introduction of the heterologous DNA into the pollen germ cell itself, the heterologous DNA then being carried down the pollen tube to the ovum along with endogenous DNA during fertilization. Another possible mechanism involves adhesion of Agrobacterium to the elongating pollen tube, whereby the bacterium is carried to the ovum by the tube, where transformation of the ovum or developing zygote occurs. The existence of several possible mechanisms necessitates the precise definition of the terms "transgenic pollen" and "transformed pollen" as set forth above.
The use of pollen as a vector is not without its problems, however. In order to effectively and efficiently obtain transgenic pollen it is necessary first to germinate the pollen grain. In an Agrobacterium based system this is required in order to allow for either transfer of the heterologous DNA from the bacteria to the pollen germ cell or for effective attachment of the bacterium of the growing pollen tube. Because of the time factor this necessitates an in vitro system for pollen germination and pollen tube growth. The cultivation of germinating pollen and pollen tubes in vitro has proved difficult, as the grains tend to rupture in the culture medium, resulting in the release and degradation of their DNA. Pollen survival has been low, and subsequent plant transformation efficiency poor. Some pollen types, so-called "dry" or "dry stigma" pollens (such as cotton pollen) are so sensitive to moisture that efforts to obtain pollen germination and sustained pollen tube growth have failed. Thus, it has proved difficult to culture and obtain transformed pollen effectively in vitro for use as a vector for producing transgenic plants. U.S. Pat. No. 5,066,594 (DeBonte et al., Nov. 19, 1991) contains a review of in vitro pollen germination methods and pollen-based methods of plant transformation, and difficulties encountered in their use. DeBonte et al. state that the consensus in the art is that calcium, boron and an osmoticum (usually sucrose) are critical components of a germination medium in order to obtain pollen germination. Col. 2, lines 10-22. DeBonte also notes that lysis of the pollen wall has been found to occur in pollen germination medium that does not contain agar. Col. 2, lines 44-49. A medium containing calcium, boron, lysine, glutamic acid and sucrose was found to give fair germination rates with pollen that had been stored for 12 hours after anthesis, but the pollen grains demonstrated poor stability and tended to burst over time on the germination medium. Col. 2, lines 50-63.
It should be noted that DeBonte was discussing exclusively germination of wet stigma pollen, and the use of liquid or semi-liquid germination media.
DeBonte proposed an aqueous "stabilization solution," to be used in conjunction with an aqueous germination medium, to permit maintenance of germinating pollen in culture for a time sufficient to allow transformation by Agrobacterium. Once germinated, the pollen would be transferred to the stabilization solution and incubated with the Agrobacterium vector to effect transformation of the pollen. DeBonte did not demonstrate that this method actually worked for transforming pollen, nor that the pollen was capable of fertilizing a receptive plant after being thus treated.
Other methods proposed for transforming pollen include microparticle bombardment (U.S. Pat. No. 5,100,792; U.S. Pat. No. 5,120,657), microinjection (U.S. Pat. No. 4,743,548) and electroporation (U.S. Pat. No. 5,629,183). The first requires elaborate and expensive equipment, while the second requires delicate manipulation of individual pollen grains. Neither method has been shown to be effective in producing viable transgenic pollen capable of fertilizing a receptive plant. The third, although effective, again requires elaborate and expensive equipment, highly knowledgeable and trained personnel and the pollen has to be exposed to aqueous conditions; a lethal environment for dry stigma pollen under normal circumstances.
A need remains for a simple, effective means of germinating and obtaining transgenic pollen in vitro, particularly dry stigma pollen, that is efficient and permits easy fertilization of receptive plants with the transgenic pollen.